Reducing agent from microorganism belonging to genus bacillus and application for same

ABSTRACT

The present invention is intended to provide a reducing agent effective for color development of meat and uses therefor. The present invention provides a reducing agent containing a heme reductase derived from a microorganism belonging to the genus  Bacillus.  Preferably, crushed bacterial cells of  Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus natto, Bacillus thuringiensis,  or  Bacillus mycoides  are used.

TECHNICAL FIELD

The present invention relates to a reducing agent derived from amicroorganism belonging to the genus Bacillus and uses thereof. Thereducing agent of the present invention is particularly useful forimproving the color tone of meat or processed meat. The presentapplication claims priority based on Japanese Patent Application No.2010-109779 filed on May 12, 2010, and the content of the patentapplication is hereby incorporated by reference herein in its entirety.

BACKGROUND ART

Meat color is an important factor for consumers to evaluate the meatquality. Bright red meat is regarded as good quality, and brown meat isregarded as stale. In this manner, the meat color markedly influencesthe consumers' willing to buy the meat and their evaluation of the meat.

The color tone of meat reflects the proportion of myoglobin derivativesin the meat. Once myoglobin is oxidized to metmyoglobin, the color tonechanges into brown, and thus the commercial value of the meat productmarkedly decreases.

In order to prevent browning of meat, processed meat such as ham andsausage is generally treated with a color development agent such as anitrate or nitrite. However, since nitrates and nitrites have acutetoxicity to cause methemogulobinemia in human, their usage is limited to70 ppm or less in terms of the residual nitrite. In addition, it is saidthat nitrous acid can react with a secondary amine to form nitrosaminewhich is a carcinogen. Therefore, from the viewpoint of safety, colordeveloping substances and color development methods which replace colordevelopment agents such as nitrates or nitrites have been searched. Forexample, a method for preventing browning through the addition ofraffinose (see Patent Document 1), a method for preventing browningthrough the addition of a Flammulina veluptipes extract (see PatentDocument 2), and a method for developing a color through the use ofcomponents contained in vegetables (see Patent Document 3) were found.However, the methods described in Patent Documents 1 and 2 cannotachieve sufficient color development effect, and the method described inPatent Document 3 uses nitrates contained in vegetables, and thus has aproblem with safety.

In addition, Patent Document 4 proposes the method of maintaining thecolor tone of meat by substituting iron in myoglobin with zinc to form azinc myoglobin-protoporphin IX complex, and Patent Documents 5 and 6propose the methods of maintaining the fresh red color of meat byaccelerating the formation of a zinc myoglobin-protoporphin IX complexthrough the use of ferrochelatase or yeast. These methods cannot act onmetmyoglobin which has been generated, and cannot achieve sufficientcolor development effect or color tone holding effect.

PRIOR ART DOCUMENT Patent Documents

Patent Document 1

Japanese Unexamined Patent Application Publication No. 2003-18976

Patent Document 2

Japanese Unexamined Patent Application Publication No. 2008-228702

Patent Document 3

Japanese Unexamined Patent Application Publication No. 2009-165445

Patent Document 4

Japanese Unexamined Patent Application Publication No. 2006-56908

Patent Document 5

Japanese Unexamined Patent Application Publication No. 2005-87058

Patent Document 6

Japanese Unexamined Patent Application Publication No. 2006-61016

SUMMARY OF THE INVENTION Problems to be Solved by the Invention

The present invention is intended to provide a reducing agent which iseffective for improving the color tone of meat or processed meat anduses thereof (for example, color tone improvement method without using acolor development agent such as a nitrite).

Means for Solving the Problem

In order to find substances which improve the color tone of meat, theinventors carried out screening mainly on microorganisms belonging tothe genus Bacillus. As a result of screening, a microorganism strainproducing substances with high meat color development effect wasidentified. As a result of further study, the substances which areexpected to be highly useful were found to exhibit reduction activity onmetmyoglobin. More specifically, the microorganism belonging to thegenus Bacillus produces the substances accelerating the development ofmeat color by metmyoglobin reduction activity. The substances exhibitreduction action on heme, and are useful in developing meat color, andalso in the applications where the reduction of heme or heme protein iseffective or necessary. For example, the substances may be used for thepurpose of reducing methemoglobin which has a structure similar tometmyoglobin.

As a result of further study, the substances derived from themicroorganism belonging to the genus Bacillus (substances exhibitingreduction action) were found to be dihydrolipoyl dehydrogenase andnitroreductase.

The present invention has been accomplished based on the above results,and includes the following aspects.

[1] A reducing agent containing a heme reductase derived from amicroorganism belonging to the genus Bacillus.

[2] The reducing agent of [1], wherein the heme is the heme ofmetmyoglobin.

[3] The reducing agent of [1], wherein the heme is the heme ofmethemoglobin. [4] The reducing agent of any one of [1] to [3], which iscomposed of crushed bacterial cells of a microorganism belonging to thegenus Bacillus.

[5] The reducing agent of any one of [1] to [4], wherein themicroorganism belonging to the genus Bacillus is a microorganismselected from the group consisting of Bacillus subtilis, Bacillusamyloliquefaciens, Bacillus natto, Bacillus thuringiensis, and Bacillusmycoides.

[6] The reducing agent of [1], wherein the heme reductase isdihydrolipoyl dehydrogenase or nitroreductase.

[7] The reducing agent of [1], which contains dihydrolipoyldehydrogenase and nitroreductase as the heme reductases.

[8] The reducing agent of [6]or [7], wherein the amino acid sequence ofthe dihydrolipoyl dehydrogenase includes the amino acid sequence of SEQID NO: 3, and the amino acid sequence of the nitroreductase includes theamino acid sequence of SEQ ID NO: 12.

[9] The reducing agent of any one of [6] to [8], wherein thedihydrolipoyl dehydrogenase and nitroreductase are recombinant proteins.

[10] A color tone improver composed of the reducing agent of any one of[1] to [9].

[11] A color tone improver composed of the reducing agent of any one of[1] to [9] and a substance which substitutes iron in the heme group ofmyoglobin with zinc.

[12] The color tone improver of [11], wherein the substance isferrochelatase.

[13] The color tone improver of any one of [10] to [12], which is usedfor improving the color tone of meat or processed meat.

[14] A color tone improver for meat or processed meat containingdihydrolipoyl dehydrogenase and/or nitroreductase.

[15] The color tone improver of any one of [10] to [14], which improvesthe color tone by color development action, color developmentacceleration action, and/or color fading preventive action.

[16] A medicine containing the reducing agent of any one of [1] to [9].

[17] The medicine of [16], which is an oral preparation.

[18] The medicine of [16], which is a parenteral preparation.

[19] A method for producing a reducing agent including the followingsteps (1) and (2):

(1) a step of culturing a microorganism belonging to the genus Bacillusproducing a heme reductase under conditions suitable for the productionof the enzyme; and

(2) a step of recovering the enzyme from the culture.

[20] The production method of [19], wherein the step (2) includes thefollowing steps:

(2-1) a step of collecting bacterial cells from the culture; and

(2-2) a step of preparing crushed bacterial cells.

[21] The production method of [19] or [20], wherein the heme is the hemeof metmyoglobin.

[22] The production method of [19] or [20], wherein the heme is the hemeof methemoglobin.

[23] The production method of any one of [19] to [22], wherein themicroorganism belonging to the genus Bacillus is selected from the groupconsisting Bacillus subtilis, Bacillus amyloliquefaciens, Bacillusnatto, Bacillus thuringiensis and Bacillus mycoides.

[24] A color tone improvement method, including subjecting meat orprocessed meat to the action of the color tone improver of any one of[10] to [15].

[25] A color tone improvement method, including subjecting meat orprocessed meat to the action of crushed bacterial cells of amicroorganism belonging to the genus Bacillus selected from the groupconsisting of Bacillus subtilis, Bacillus amyloliquefaciens, Bacillusnatto, Bacillus thuringiensis, and Bacillus mycoides.

[26] A prophylactic or therapeutic method using the reducing agent ofany one of [1] to [9] for a disease associated with or caused by one ormore clinical conditions or symptoms selected from blood circulationdisorder and hypoxia or hypoxemia.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the absorption spectra of the solutions containing thecrushed bacterial cell extract of a microorganism belonging to the genusBacillus (or its heat treated extract) and freeze-dried ham powder.

FIG. 2 shows the changes over time in the absorbance at 580 nm (A₅₈₀) ofsolutions each containing the crushed bacterial cell extract of amicroorganism belonging to the genus Bacillus and freeze-dried hampowder.

FIG. 3 shows the metmyoglobin reductase activity in the freeze-driedpowder of the crushed bacterial cell extracts of microorganismsbelonging to the genus Bacillus.

FIG. 4 shows the elution pattern and metmyoglobin reductase activityobtained by DEAE chromatography during purification of metmyoglobinreductase.

FIG. 5 shows the elution pattern and metmyoglobin reductase activityobtained by hydroxyapatite chromatography during purification ofmetmyoglobin reductase.

FIG. 6 shows the elution pattern and metmyoglobin reductase activityobtained by gel filtration chromatography after purification ofmetmyoglobin reductase.

FIG. 7 shows the result of SDS-PAGE of the gel filtration fraction withhigh specific activity.

FIG. 8 shows the comparison of specific activity of the samples obtainedduring purification.

FIG. 9 shows the result of meat color development test using a purifiedenzyme (dihydrolipoyl dehydrogenase: DLD). The samples with differentenzyme amounts were compared.

FIG. 10 shows the result of meat color development test using a purifiedenzyme (DLD). The samples with or without NADH were compared.

FIG. 11 shows the result of rate assay (substrate-saturation curve) of apurified enzyme (DLD).

FIG. 12 shows the result of rate assay ([s]/v to [s] plot) of thepurified enzyme (DLD).

FIG. 13 shows the optimal pH of the purified enzyme (DLD).

FIG. 14 shows the pH stability of the purified enzyme (DLD).

FIG. 15 shows the optimal temperature of the purified enzyme (DLD).

FIG. 16 shows the thermal stability of the purified enzyme (DLD).

FIG. 17 shows the reactivity of the purified enzyme (DLD) to NADH andNADPH.

FIG. 18 shows the influences of various cations on the activity of thepurified enzyme (DLD).

FIG. 19 shows the metmyoglobin reduction activity of recombinant DLD(without His tag).

FIG. 20 shows the metmyoglobin reduction activity of recombinant DLD(with His tag).

FIG. 21 shows the result of meat color development test usingrecombinant DLD. The R value, G value, and B value of the negativecontrol (left) were 178, 104, and 102, respectively, and those of thepurified recombinant DLD (right) were 210, 104, and 114, respectively.

FIG. 22 shows the result of meat color development test usingrecombinant DLD. The R value, G value, and B value of sample 1 were 201,117, and 123, respectively, those of sample 2 were 185, 121, and 105,those of sample 3 were 217, 97, and 93, those of sample 4 were 160, 108,and 83, and those of sample 5 were 156, 84, and 65, respectively.

FIG. 23 shows the result of meat color development test usingrecombinant DLD. The R value, G value, and B value of sample 1 were 168,122, and 106, respectively, those of sample 2 were 185, 152, and 145,those of sample 3 were 172, 123, and 119, those of sample 4 were 187,153, and 144, and those of sample 5 were 173, 148, and 123,respectively.

FIG. 24 shows the elution pattern and metmyoglobin reductase activityobtained by phenyl chromatography during purification of a meat colordeveloping enzyme other than DLD.

FIG. 25 shows the elution pattern and metmyoglobin reductase activityobtained by hydroxyapatite chromatography during purification of a meatcolor developing enzyme other than DLD.

FIG. 26 shows the elution pattern and metmyoglobin reductase activityobtained by Cu affinity chromatography during purification of a meatcolor developing enzyme other than DLD.

FIG. 27 shows the result of SDS-PAGE of the fractions obtained byhydroxyapatite chromatography and Cu affinity chromatography.

FIG. 28 shows the activities of recombinant malate dehydrogenase (MDH)and recombinant nitroreductase (Putative NAD(P)H nitroreductase: yodC).From left to right in this order, yodC/pET20b/BL21 (DE3pLysS) (Bacillussubtilis), yodC/pET20b/BL21 (DE3pLysS) (Bacillus natto), pET20b/BL21(DE3pLysS) (empty vector), MDH/pET20b/BL21 (DE3pLysS) (Bacillussubtilis), and MDH/pET20b/BL21 (DE3pLysS) (Bacillus natto).

FIG. 29 shows the result of SDS-PAGE of recombinant MDH and recombinantyodC. From left to right in this order, marker, MDH/pET20b/BL21(DE3pLysS) (Bacillus subtilis), yodC/pET20b/BL21 (DE3pLysS) (Bacillussubtilis), pET20b/BL21 (DE3pLysS), MDH/pET20b/BL21 (DE3pLysS) (Bacillusnatto), yodC/pET20b/BL21 (DE3pLysS) (Bacillus natto), and marker.

FIG. 30 shows the result of meat color development test using purifiedrecombinant yodC and recombinant MDH.

FIG. 31 shows the optimal pH of recombinant yodC.

FIG. 32 shows the pH stability of recombinant yodC.

FIG. 33 shows the optimal temperature of recombinant yodC.

FIG. 34 shows the thermal stability of recombinant yodC.

FIG. 35 shows the reactivity of recombinant yodC to NADPH.

FIG. 36 shows the influences of various cations on the activity ofrecombinant yodC.

FIG. 37 shows the comparison of reactivity of DLD and yodC to potassiumferricyanide.

FIG. 38 shows the comparison of reactivity of DLD and yodC to myoglobin.

DESCRIPTION OF EMBODIMENTS

(Terms)

In the present description, the term “heme” means a complex composed ofan iron atom and porphyrin (iron-porphyrin complex). The term “hemeprotein” is the generic name of protein including heme. The term “hemereductase” means a protein exhibiting reduction activity to the ironatom in heme. The degree of the activity is not particularly limited.Typically, a heme reductase exhibits activity reducing the methocompound of heme protein. With the focus on the activity, heme reductasemay be called heme protein reductase.

In the present description, “metmyoglobin reductase” refers to a proteinexhibiting activity reducing metmyoglobin, which is a myoglobinderivative. The intensity (degree) of the activity is not particularlylimited. Accordingly, even if reduction activity of an enzyme onmetmyoglobin is weaker than other activity of the enzyme, the enzyme isincluded in the “metmyoglobin reductase” referred herein.

In the present description, “color tone improver” refers to a substanceor composition used for the improvement of “color tone”, the formationof which is contributed by a metalloporphyrin complex. Examples of themetalloporphyrin complex include a copper porphyrin complex, a cobaltporphyrin complex, and an iron porphyrin complex. But themetalloporphyrin complex is not limited to these examples, as long asthe metal in the complex can be reduced. Preferred examples of themetalloporphyrin complex include an iron porphyrin complex, and examplesof the composition containing the iron porphyrin complex include hemeprotein. Examples of the composition rich in heme protein include meator processed meat.

The color tone improver of the present invention improves the color toneof the target by color development action, color developmentacceleration action, and/or color fading prevention action. For example,the color tone improver of the present invention can reduce the metal ina metalloporphyrin complex to improve the color tone. Alternatively, thecolor tone improver can prevent oxidation of a dye composed of ametalloporphyrin complex to maintain the color tone of the dye, and thusimprove the color tone. A preferred target for which the color toneimprover of the present invention is used is meat or processed meat.More specifically, according to a preferred embodiment, the color toneimprover of the present invention is used to color development, colortone maintenance, or color fading prevention of meat or processed meat.The term “color development of meat” means the development of a redcolor tone characteristic to meat or processed meat.

1. Reducing Agent Derived From the Genus Bacillus

A first aspect of the present invention relates to a reducing agent. Thereducing agent of the present invention includes a heme reductaseproduced by Bacillus as an active ingredient. As shown by thebelow-described examples, as a result of the large-scale screening bythe inventors, it was found that Bacillus subtilis, Bacillusamyloliquefaciens, Bacillus natto, Bacillus thuringiensis, and Bacillusmycoides, which are microorganisms belonging to the genus Bacillus,produce polypeptides having high metmyoglobin reduction activity. On thebasis of this finding, a preferred embodiment of the present inventionuses a metmyoglobin reductase produced by any of these microorganisms.These microorganisms are available from, for example, public storageinstitutions such as NBRC (Biological Resource Center, NationalInstitute of Technology and Evaluation), JCM (RIKEN BioResource Center),and. ATCC (American Type Culture Collection). Bacillus natto iscommercially and readily available. In addition, Bacillus natto isavailable from Miyagino Bacillus Natto Manufacturer.

The reducing agent of the present invention contains the activeingredient (polypeptide), and may further contain, for example, anexcipient, a buffering agent, a suspending agent, a stabilizer, a pHcontrolling agent, a preservative, an antiseptic, a perfume, athickener, an oil or fat, a brightener, a binder, a binding reinforcer,an emulsification stabilizer, or a normal saline solution. Examples ofthe excipient include starch, dextrin, maltose, trehalose, lactose,D-glucose, sorbitol, D-mannitol, white sugar, and glycerol. Examples ofthe buffering agent include phosphates, citrates, and acetates. Examplesof the stabilizer include propylene glycol and ascorbic acid. Examplesof the pH controlling agent include organic acids such as itaconic acid,succinic acid, tartaric acid, fumaric acid, citric acid, malic acid,adipic acid, gluconic acid, pyrophosphoric acid, acetic acid, lacticacid, α-ketoglutaric acid, and phytic acid, and salts of these organicacids; inorganic acids and salts of inorganic acids such as carbonates;acidic amino acids such as aspartic acid and glutamic acid; and basicamino acids such as arginine, lysine, and histidine. Examples of thepreservative include phenol, benzalkonium chloride, benzyl alcohol,chlorobutanol, and methylparaben. Examples of the antiseptic includeethanol, benzalkonium chloride, paraoxybenzoic acid, and chlorobutanol.Examples of the perfume include animal perfumes such as musk, civet,castoreum, and ambergris; vegetable perfumes such as anise essentialoil, angelica essential oil, ylang ylang essential oil, iris essentialoil, fennel essential oil, orange essential oil, Cananga essential oil,caraway essential oil, cardamom essential oil, guaiacum essential oil,cumin essential oil, Lindera umbellata essential oil, cinnamon essentialoil, cinnamon essential oil, geranium essential oil, copaiba balsamessential oil, coriander essential oil, perilla essential oil, cedarwoodessential oil, citronella essential oil, jasmine essential oil, gingerglass essential oil, cedar essential oil, spearmint essential oil,peppermint essential oil, star anise essential oil, tuberose essentialoil, clove essential oil, neroli essential oil, wintergreen essentialoil, tolu balsam essential oil, patchouli essential oil, rose essentialoil, palmarosa essential oil, Japanese cypress essential oil, hibaessential oil, sandal wood essential oil, petitgrain essential oil,, baylaurel essential oil, vetiver essential oil, bergamot essential oil,Peru balsam essential oil, bois de rose essential oil, linaloolessential oil, mandarin essential oil, eucalyptus essential oil, limeessential oil, lavender essential oil, linaloe essential oil, lemonglass essential oil, lemon essential oil, rosemary essential oil, andJapanese peppermint essential oil; and other synthetic perfumes.Examples of the thickener include natural polymer or starch or cellulosenatural polymer derivatives. Examples of the natural polymer includeseaweed extracts such as fucoidan and carrageenan, seed extracts such asguar gum, resin-like slimes such as gum arabic, andmicroorganism-produced slimes such as xanthan gum. Examples of thestarch or cellulose natural polymer derivatives include starch-based(for example, starch phosphate) or cellulose-based (for example, methylcellulose-based) natural polymer derivatives. Examples of the oil andfat include avocado oil, linseed oil, almond oil, fennel oil, perillaoil, olive oil, orange oil, orange roughy oil, cacao butter, camomileoil, carrot oil, cucumber oil, coconut oil, sesame oil, rice oil,safflower oil, shea butter, liquid shea butter, soybean oil, camelliaoil, corn oil, rapeseed oil, persic oil, castor oil, sunflower oil,grape seed oil, cottonseed oil, peanut oil, turtle oil, mink oil, eggyolk oil, palm oil, palm kernel oil, Rhus succedanea fruit wax, coconutoil, beef tallow, and lard. These oils and fats may be modified by, forexample, hydrogenation, fractionation, or interesterification. Examplesof the brightener include waxes (vegetable or animal) such as beeswax,carnauba wax, whale wax, lanolin, liquid state lanolin, reduced lanolin,hard lanolin, candelilla wax, montan wax, shellac wax, rice wax,squalene, squalane, and pristane; and mineral oils such as liquidparaffin, vaseline, paraffin, ozokerite, ceresin, and microcrystallinewax. Examples of the binder include soybean protein, egg protein, milkprotein, blood protein, casein, starch, and transglutaminase. Examplesof the binding reinforcer include polymerized phosphates. Examples ofthe emulsification stabilizer include casein sodium. Examples of theother additive include natural fatty acids such as lauric acid, myristicacid, palmitic acid, stearic acid, behenic acid, oleic acid, linoleicacid, linolenic acid, docosahexaenoic acid, eicosapentaenoic acid,1,2-hydroxy stearic acid, undecylenic acid, tall oil, and lanolin fattyacid; and synthetic fatty acids such as isononanoic acid, caproic acid,2-ethyl butanoic acid, isopentane acid, 2-methylpentanoic acid, 2-ethylhexanoic acid, and isopentanoic acid.

According to an embodiment, the reducing agent of the present inventionis composed of crushed bacterial cells of the microorganism according tothe present invention which produces polypeptide. More specifically, thereducing agent according to this embodiment contains the crushedbacterial cells of the specified microorganism. A crushed bacterial cellsolution (normally obtained by a procedure including culture of themicroorganism, collection of the microorganism, and crushing of thebacterial cells) may be used as the crushed bacterial cells.Alternatively, the crushed bacterial cell solution may be subjected tofurther treatment (for example, purification treatment, freezingtreatment, drying treatment, or addition of other components) before theuse as crushed bacterial cells.

As a result of the study by the inventors, it was found that the activeingredients derived from a microorganism belonging to the genus Bacillus(substances exhibiting reduction action, and effective particularly atimproving the color tone of meat) are dihydrolipoyl dehydrogenase (DLD)and nitroreductase (yodC) (see the below-described examples). Therefore,one aspect of the present invention provides a reducing agent containingdihydrolipoyl dehydrogenase or nitroreductase derived from amicroorganism belonging to the genus Bacillus as heme reductase.According to a preferred embodiment, the reducing agent contains both ofdihydrolipoyl dehydrogenase and nitroreductase. The amino acid sequenceof dihydrolipoyl dehydrogenase is shown in SEQ ID NO: 3, and that ofnitroreductase is shown in SEQ ID NO: 12.

The identification of effective heme reductases and identification oftheir amino acid sequences have allowed the use of enzymes prepared by agenetic engineering method. Therefore, one aspect of the presentinvention uses an enzyme which has been prepared by a geneticengineering method, more specifically a heme reductase composed of arecombinant protein. Specifically, a reducing agent containingrecombinant dihydrolipoyl dehydrogenase and/or recombinantnitroreductase as active ingredients is provided. Recombinant proteinrefers to an artificially prepared protein by a gene recombinationtechnique.

2. Applications of Reducing Agent

A second aspect of the present invention relates to the applications ofthe reducing agent of the present invention. The applications providedby the present invention are broadly divided into the improvement of thecolor tone and other uses. The former application, more specifically theuse as a color tone improver is particularly important. The targets ofcolor tone improvement by the present invention are those involved witha metalloporphyrin complex in the formation of the color tone. Preferredexamples of the target include meat and processed meat. The color toneof meat reflects the proportion of the myoglobin derivatives containedin the meat. As described above, the reducing agent of the presentinvention contains a heme reductase (preferably metmyoglobin reductase)as an active ingredient. Accordingly, when the reducing agent of thepresent invention is allowed to act on meat, metmyoglobin in the meat isreduced to form reduced myoglobin. The reduced myoglobin is oxygenatedto be converted to oxymyoglobin which shows a bright red color tone.Upon the action of the reducing agent of the present invention, themetmyoglobin level in meat or processed meat decreases, and at the sametime oxymyoglobin is formed, which results in the improvement of thecolor tone. In addition, the oxidation of reduced myoglobin oroxymyoglobin is prevented, and as a result of this, color fading of themeat is prevented. In this manner, the reducing agent of the presentinvention will exhibit the effects of color development and color tonemaintenance, or prevention of color fading. The target of the colorfading prevention effect is a metalloporphyrin complex, and the complexis not particularly limited as long as the metal in the complex isoxidizable. A preferred metalloporphyrin complex is an iron porphyrincomplex. Accordingly, a preferred target of the color fading preventioneffect is a heme protein containing an iron porphyrin complex. Mostpreferred targets are reduced myoglobin or oxymyoglobin, and meat orprocessed meat containing reduced myoglobin and/or oxymyoglobin.

When the reducing agent of the present invention is used for the colortone improvement of meat or processed meat (more specifically, the colortone improvement method using the reducing agent of the presentinvention as a color tone improver), meat or processed meat is treatedby the reducing agent of the present invention. The treatment conditionsare set to be generally suitable for the action of the metmyoglobinreductase composing the reducing agent (preferably optimal conditions).The preferred treatment conditions are readily specified based on thepreliminary experiment using the meat or processed meat to be treated.Specific examples of the treatment method (the use of a solution ofcrushed bacterial cells for the improvement of meat color tone).Firstly, a solution of crushed bacterial cells of a certainmicroorganism (a microorganism belonging to the genus Bacillus producingmetmyoglobin reductase) is prepared, and its pH is adjusted to about5.5, thereby reproducing the pH in meat. Subsequently, meat is exposedto the solution at 4° C. The exposure is usually achieved by injectionof a suspension into block meat, followed by tumbling, or blending ofminced meat with a suspension. When the exposure is achievedappropriately, the suspension thoroughly penetrates into meat. Thetreatment temperature may be near 40° C. for developing a color, but ispreferably 4° C. or near 4° C. in consideration of meat quality.

The type of meat to be treated is not limited. As described above, thecolor tone of meat reflects the proportion of the myoglobin derivativescontained in meat. The reducing agent of the present inventioninfluences the proportion of the myoglobin derivatives in meat, andaccelerates the color development. Accordingly, the present invention isapplicable to meat or processed meat in general containing myoglobin.Specific examples of the target to be treated include meat such as pork,beef, and chicken, processed meat thereof, fish meat such as tuna,bonito, and salmon, and processed fish meat thereof. A preferred targetis meat showing a red color. Processed meat as a target to be treated isnot particularly limited as long as it is a food produced from meat.Examples of the processed meat include raw ham, sausage, and loin roll.The form of the meat or processed meat to be treated is not particularlylimited either, and may be selected from, for example, block meat orminced meat, according to the intended use.

According to one aspect of the present invention, the reducing agent ofthe present invention is used in combination with a substance whichsubstitutes iron in the heme group of myoglobin with zinc (hereinafterreferred to as “iron-zinc substituting substance”). When the reducingagent of the present invention is used in combination with a substancewhose action is different from the reducing agent, the color tone isfurther improved owing to multiple effect. In particular, thecombination results in the maintenance of good color tone and preventionof color fading. Examples of the iron-zinc substituting substanceinclude ferrochelatase (more specifically, see Japanese UnexaminedPatent Application Publication No. 2006-61016). Ferrochelatase ispresent in animal tissues (particularly viscera), plant tissues (forexample, mushrooms, bean sprouts, and pea bean), yeasts (for example,bread yeast, beer yeast, sake yeast, wine yeast, and shochu yeast), andbacteria. Ferrochelatase extracted from these natural products may beused. In addition, ferrochelatase is rich in mitochondria fractions, sothat the use of mitochondria fractions are particularly preferred. Theiron-zinc substituting substance may be a Saccharomyces yeast (forexample, beer yeast, bread yeast, sake yeast, or shochu yeast) (morespecifically, see Japanese Unexamined Patent Application Publication No.2005-87058).

This embodiment is characterized in that it uses the reducing agent ofthe present invention in combination with the iron-zinc substitutingsubstance. Typically, the color tone improver of the present inventionis provided as a combination agent composed of the reducing agent of thepresent invention and the iron-zinc substituting substance. On the otherhand, for example, the color tone improver of the present invention maybe provided in the form of a kit composed of the reducing agent of thepresent invention (first component) and an agent (second component)containing the iron-zinc substituting substance. In this case, thetarget to be treated (meat or processed meat) is treated simultaneouslyor consecutively by the first and second components. The term“simultaneously” does not mean strict simultaneousness. Accordingly, theconcept “simultaneously” herein include the case wherein two componentsare used without time difference, such as the use of these componentsafter mixing them, and the case wherein two components are usedsubstantially without time difference, such as the use of one componentimmediately after the use of the other component.

When the reducing agent of the present invention is used as a color toneimprover, the color tone improver may contain, in addition to the activeingredient (polypeptide) and additives (for example, an excipient, abuffering agent, a suspending agent, a stabilizer, a pH controllingagent, a preservative, an antiseptic, a perfume, a thickener, an oil orfat, a brightener, a binder, a binding reinforcer, an emulsificationstabilizer, or a normal saline solution), a condiment, a spice, amasking agent, a softener, or the like. Examples of the condimentinclude soy sauce, miso, vinegar, sake, mirin (Japanese sweet rice winefor cooking), salt, soup stock of bonito or kelp, meat extract, orvegetable extract. Examples of the spice include pepper, laurel, thyme,clove, oregano, star anise, Japanese pepper, sage, parsley, nutmeg,mustard, ginger, cinnamon, basil, paprika, rosemary, spearmint, lemonglass, tarragon, chervil, cardamom, cumin, coriander, dill, fennel,marjoram, and allspice. Examples of the masking agent includesaccharides such as sucrose and cyclodextrin; and herbs such as clove,allspice, laurel, cinnamon, and nutmeg. Examples of the softener includeproteolytic enzymes such as protease, trypsin, chymotrypsin, papain,bromelain, and ficin.

The reducing agent of the present invention may be used for meat orother targets. The reducing agent of the present invention may be usedfor the purpose of reduction or oxidation prevention (including colorfading prevention) of a composition containing a heme protein other thanmyoglobin (for example, hemoglobin).

The reducing agent of the present invention is expected to be used forthe measurement of the hemoglobin concentration and the treatment ofhemoglobinemia. More specifically, the reducing agent of the presentinvention is useful as an active ingredient of reagents and medicines.At present, the cyan-methemoglobin method is frequently used formeasuring the hemoglobin concentration of blood. According to themethod, methemoglobin is subjected to the action of a mixture ofpotassium ferricyanide and potassium cyanide to form cyan methemoglobin,and measured by colorimetric determination. The reducing agent of thepresent invention can be used in place of the method for measuring theblood methemoglobin level or total methemoglobin content.

Methemogulobinemia is caused by oxygen deficiency in the body due toexcessive accumulation of methemoglobin for some reason. The mosteffective treatment for methemogulobinemia is believed to be theintravenous injection of methylene blue. However, whenmethemogulobinemia is complicated with cyanide poisoning, methylene bluecannot be used because it accelerates cyanide poisoning. Examples ofother treatment include oral administration and intravenous injection ofascorbic acid (ascorbic acid may be administered in combination withriboflavin), but these methods are not so effective. The reducing agentof the present invention allows a new treatment strategy which replacesthese conventional treatment methods. The treatment using the reducingagent of the present invention may be used for patients to whommethylene blue cannot be administered (for example, patients havingcyanide poisoning). In addition, methylene blue is ineffective for thoselack in glucose-6-phosphate dehydrogenase (G6PD). The patients withdisorders in the pentose phosphate pathway such as G6PD deficiency willnot respond to the approach, and must receive urgent exchangetransfusion.

G6PD deficiency is one of most common disorders in the world. About 10%male of black male in the United States have the disorder. Many patientsare inhabitants in Africa and the Mediterranean. Accordingly, asubstantial proportion of subjects are at risk of developing (oxidative)drug-induced methemogulobinemia. The administration of methylene blue tothese patients is ineffective (because G6PD deficiency in them willcause NADPH deficiency), and can be counterproductive. The reducingagent of the present invention is expected to be effective for thesepatients.

The reducing agent of the present invention can exhibit pharmacologicalaction and physiological action such as the reduction of burden on theheart caused by the disorders of cardiac rate, blood pressure, andcardiac output, and acceleration of metabolism in tissues. The medicinecontaining the reducing agent of the present invention is expected to beused as a tolerance enhancer for enhancing the body tolerance underphysiological conditions with high oxygen demand of tissues, such as theenvironments with hard work and exercise. In addition, the medicine ofthe present invention may be used for prevention or treatment of cardiacfailure, cardiac myopathy, myocarditis, myocardial infarction,pericarditis, perimyocarditis, transient ischemia attack, coronary heartdisease, congenital anomaly with right to left arteriovenous shunt(vitia), tetralogy/pentalogy of Fallot, Eisenmenger's syndrome, shock,peripheral ischemia, arterial occlusive disease (AOD), peripheral AOD(pAOD), carotid artery stenosis, renal artery stenosis, microcirculatorydisorders in the brain (arteriocapillary sclerosis), bleeding in thebrain, cerebral vein blood clots and intracranial venous sinusthrombosis, angiodysplasia, subarachnoid hemorrhage, vascular dementia,Biswanger's disease, subcortical arteriosclerotic encephalopathy,multiple cortical infarction accompanied by embolization, vasculitis,diabetic retinopathy, prognosis of anemia due to various causes (forexample, aplastic anemia, myelodysplastic syndrome, polycythemia vera,anemia megaloblastic, hypoferric anemia, renal anemia, sphaerocytosis,and haemolytic anemia), thalassemia, hemoglobinopathy,glucose-6-phosphate dehydrogenase deficiency, transfusion onset, rhesusincompatibility, malaria, valvuloplasty, acute post hemorrhagic anemia,hypersplenism, lung fibrosis, emphysema, lung oedema: ARDS, IRDS, orrecurrent lung emphysema, burn, angina pectoris, ischemic disease suchas hibernation, blood circulation disorder, hypoxia, or hypoxemia, orprevention or treatment of diseases accompanied by or caused by any ofthese diseases or symptoms.

In order to prevent ischemic cell damage through the supply of oxygen tothe ischemic tissues (and to protect tissues from reperfusion injury),the medicine of the present invention may be administered before,during, and/or after the occurrence of ischemic event. In combinationwith the administration of the medicine of the present invention, avasoactive oxygen carrier (for example, oxygen carrier based onhemoglobin) may be administered. For example, in order to achieveintended treatment effect in mammal against acute ischemia and followingreperfusion and release of free radical caused by surgicalrevascularization (for example, percutaneous transluminal coronaryrevascularization), transplantation, acute myocardial infarction, orangioplasty (for example, percutaneous transluminal coronaryangioplasty), the combination of the medicine of the present invention,a vasoactive carrier (for example, oxygen carrier based on hemeprotein), and gaseous nitric oxide may be administered, or thecombination of the medicine of the present invention and a vasoactivecarrier may be administered after the administration of gaseous nitricoxide. The mammal treated by the method described herein may haveischemic heart disease, acute ischemic condition (for example,myocardial infarction, cerebral apoplexy, or renal ischemia), orangiospasm in the organ (for example, brain, heart, kidney, liver, orgastrointestinal tract) before treatment.

The medicine of the present invention may be used for treating hypoxemictissues in vertebrate animals caused by various factors including thedecrease in the red blood cell flow rate in a part of or throughout thecirculating system, anemia, and cerebral apoplexy. In addition, themedicine of the present invention may be prophylactically used for thepurpose of preventing oxygen deficiency in tissues of vertebrateanimals. Furthermore, the medicine of the present invention may be usedfor the purpose of treating or preventing hypoxia caused by partialblock in partial arterial obstruction or minute circulation. Regardingthe administration of hemoglobin, see U.S. patent application Ser. No.08/409,337.

The dose and administration period of the medicine of the presentinvention are not particularly limited, and may be appropriatelyselected in accordance with the administration form, age, body weight,symptoms, and the like.

The subject of the medicine of the present invention is not limited, andinclude humans, mammals other than humans (including pet animals,livestock, and experimental animals; specific examples include monkeys,mice, rats, guinea pigs, hamsters, monkeys, bovines, pigs, goats, sheep,horses, chickens, sheep, whales, dolphins, dogs, and cats). Thetreatment subject may have euvolemia, hypervolemia, or hypovolemiabefore, during, and/or after administration of the medicine of thepresent invention.

The administration form of the medicine of the present invention is notparticularly limited. The medicine may be orally or parenterallyadministered. Examples of the “parenteral” administration used herein isnot particularly limited, and examples thereof include intravenous,intramuscular, intraarterial, intraspinal, intracystic, intraorbital,intracardiac, intradermal, intraperitoneal, transtracheal, hypodermic,subepidermal, intracapsular, subcapsular, subarachnoid, intraspinal, andintrasternal infusion and injection. The administration form ispreferably intravenous injection.

Examples of the preparation suitable for oral administration includetablets, capsules, powders, fine grains, granules, solutions, andsyrups. Examples of the preparation suitable for parenteraladministration include injections, suppositorys, inhalants, and patches.The medicine of the present invention may contain, as necessary, apharmacologically and pharmaceutically acceptable additive. Examples ofpharmacologically and pharmaceutically acceptable additive includeexcipients, disintegrating agents or disintegrating aids, binders,lubricants, coating agents, dyes, diluents, bases, solubilizers orsolubilizing agents, isotonizing agents, pH adjusting agents,stabilizers, propellants, and adhesives.

The preparation suitable for oral administration or parenteraladministration may contain additives such as excipients such as glucose,lactose, D-sorbitol, D-mannitol, starch, kaolin, xylitol, dextrin, cornstarch, potato starch, hydroxypropyl cellulose, or crystallinecellulose; disintegrating agents or disintegrating aids such ascarboxymethyl cellulose, starch, or carboxymethyl cellulose calcium;binders such as hydroxypropyl cellulose, hydroxypropyl methyl cellulose,polyvinyl pyrrolidone, or gelatin; lubricants such as light anhydroussilicic acid, synthetic aluminium silicate, stearic acid, calciumstearate, magnesium stearate, or talc; coating agents such ashydroxypropyl methyl cellulose, white sugar, polyethylene glycol, ortitanium oxide; bases such as vaseline, liquid paraffin, polyethyleneglycol, gelatin, kaolin, glycerin, purified water, or hard fat;propellants such as Freon, diethyl ether, or compressed gas; adhesivessuch as sodium polyacrylate, polyvinyl alcohol, methyl cellulose,polyisobutylene, or polybutene; additives for preparations such as basecloth including cotton cloth or plastic sheet. Examples of thepreparation suitable for injection include solubilizers or solubilizingagents suitable as a component of an aqueous injection or an injectionwhich is dissolved before use such as distilled water, normal salinesolution, or propylene glycol; isotonizing agents such as glucose,sodium chloride, D-mannitol, and glycerin; pH controlling agents such asorganic acids (for example, itaconic acid, succinic acid, tartaric acid,fumaric acid, citric acid, malic acid, adipic acid, gluconic acid,pyrophosphoric acid, lactic acid, α-ketoglutaric acid, or phytic acid)or salts of these organic acids, inorganic acids (for example, carbonicacid) or salts of these inorganic acids, acidic amino acids (forexample, aspartic acid or glutamic acid), basic amino acids (forexample, arginine, lysine, or histidine); and soothing agents such aslidocaine.

One of typical targets of the medicine of the present invention ishemoglobin. The hemoglobin is not particularly limited, and may benatural (unmodified) hemoglobin, genetically modified hemoglobin, orchemically modified hemoglobin treated by chemical reaction such asintramolecular or intermolecular crosslinking, polymerization, oraddition of chemical group (for example, polyalkylene oxide,polyethylene glycol, superoxide dismutase, or other adduct). Themedicine of the present invention may be used to a heme protein otherthan the hemoglobin, and also to a metalloporphyrin complex having asimilar structure to the heme protein.

3. Method for Producing Reducing Agent

Another aspect of the present invention provides a method for producingthe reducing agent of the present invention. The production method ofthe present invention includes a step of culturing a heme reductase,preferably a microorganism belonging to the genus Bacillus producing ametmyoglobin reductase under conditions suitable for the production ofthe enzyme (step (1)), and a step of recovering the enzyme from theculture (step (2)).

The microorganism belonging to the genus Bacillus in the step (1) ispreferably a microorganism selected from the group consisting ofBacillus subtilis, Bacillus amyloliquefaciens, Bacillus natto, Bacillusthuringiensis, and Bacillus mycoides. The culture method and cultureconditions are not particularly limited as long as the intended enzymeis produced. More specifically, the culture method and cultureconditions suitable for the culture of the microorganism to be used areappropriately established with the proviso that a polypeptide exhibitingheme reduction activity is produced. As examples of the cultureconditions, the culture medium, incubation temperature, and incubationtime are described below.

The culture medium is selected from those that allows the growth of themicroorganism to be used. The medium may include a carbon source such asarabinose, xylose, glucose, fructose, galactose, sucrose, gentiobiose,soluble starch, glycerin, dextrin, molasses, or an organic acid, anitrogen source such as ammonium sulfate, ammonium carbonate, ammoniumphosphate, ammonium acetate, corn gluten meal, soybean powder, casaminoacid, ground coffee, cottonseed oil cake, peptone, yeast extract, cornsteep liquor, casein hydrolysate, bran, or meat extract, and aninorganic salt such as a potassium salt, a magnesium salt, a sodiumsalt, a phosphate, a manganese salt, an iron salt, or a zinc salt. Inorder to accelerate the growth of the microorganism to be used, theculture medium may further contain, for example, a vitamin or an aminoacid. The microorganism is cultured under aerobic conditions, whereinthe pH of the culture medium is, for example, about 3 to 8, preferablyabout 5 to 7, the incubation temperature is normally about 10 to 50° C.,preferably about 25 to 35° C., the incubation period is about 1 to 15days, and preferably about 3 to 7 days. Examples of the culture methodinclude stationary culture, shaking culture, and aerobic submergedculture using a jar fermenter.

After culturing under the above-described conditions, the intendedenzyme is recovered from the culture (step (2)). Typically, after theoperation of collecting the bacterial cells from the culture (step(2-1)), crushed bacterial cells are prepared (step (2-2)). Thecollection of the bacterial cells may be achieved by, for example,centrifugation or filtration. When a solid medium is used, the solidcomponents other than the bacterial cells are preferably removed inadvance. Preparation of the crushed bacterial cells may be achieved bymechanical crushing using a French press or Dyno-Mill, ultrasonication,or freezing crushing. When the bacterial cells can be crushed during thefreezing treatment, drying treatment, or freeze-drying treatment, thestep of crushing the bacterial cells may be unnecessary. The preparedcrushed bacterial cells are used as the reducing agent of the presentinvention after additional treatment or untreated (more specifically,without special treatment). Examples of the “additional treatment”include concentrated (for example, concentration using an ultrafilter),purification (for example, salting-out or various chromatography),addition of other components, dilution, and drying. The additionaltreatment may include two or more kinds of treatment. The final statemay be liquid or solid (including powder).

EXAMPLES

In order to find the substance which improves the color tone of meat,screening was carried out mainly on microorganism belonging to the genusBacillus. The results of the experiments on the microorganism strains,which had been regarded as having high usefulness based on the result ofscreening, are described below.

1. Preparation of freeze-dried powder of Bacillus subtilis, Bacillusamyloliquefaciens, Bacillus natto, Bacillus thuringiensis, Bacillusmycoides

10 mL of the liquid medium shown in Table 1 was poured into a test tube,and sterilized at 120° C. for 20 minutes. As preculture, 1 Öse portionof Bacillus subtilis strain JCM1465 (=strain ATCC6051, IAM12118, andIFO13719), Bacillus amyloliquefaciens strain NBRC15535 (=strainATCC23350), Bacillus natto, Bacillus thuringiensis strain NBRC13865(=strain ATCC13366), and Bacillus mycoides strain IAM1190 (=strainIFO3039) each was inoculated in the test tube, and cultured overnight at30° C. under shaking at 300 rpm.

TABLE 1 % (w/v) Glucose 2 Yeast extract 1 Peptone 2

In the next place, 100 mL of the liquid medium shown in Table 1 waspoured into a 300-mL conical flask, sterilized at 120° C. for 20minutes, and used as the main culture medium. As the main culture, 1 mLof the preculture solution was inoculated, and cultured overnight at 30°C. under shaking at 200 rpm. The culture solution was centrifuged at5,000 rpm for 5 minutes, and thus obtaining bacterial cells. Thebacterial cells thus obtained were washed once with 30 mL of 20 mMphosphoric acid buffer (pH 7.5), and suspended in 30 mL of 20 mMphosphoric acid buffer (pH 7.5). The resultant suspension was frozen at−40° C. for 24 hours. Subsequently, freeze-drying (20° C., 24 hours) wascarried out, and thus obtaining a freeze-dried powder (the bacterialcells were crushed by freeze-drying).

2. Meat Color Development Test 1

0.1 g of the freeze-dried powder was dissolved in 50 μL of 0.5 Mphosphoric acid buffer (pH 5.5). Subsequently, 2 g of minced ham wasmixed with the above-described powder solution and 50 μL of 4% (w/v)myoglobin, hermetically sealed, and allowed to stand at 4° C. for 17hours. The change of the red color of the meat (degree of colordevelopment) was visually observed. As control test, a sample withoutthe powder solution and another sample containing the powder solutionboiled for 10 minutes were subjected to the same test. The results areshown in Table 2. The result of the treatment of Pichia farinose strainIAM12223 (=strain IFO0465, JCM1634) with a freeze-dried powder solutionis also shown (data of untreated sample alone).

TABLE 2 Degree of color Sample development Bacillus subtilis Untreated++ Heat treated − Bacillus amyloliquefaciens Untreated + Heat treated −Bacillus natto Untreated ++ Heat treated − Bacillus thuringiensisUntreated + Heat treated − Bacillus mycoides Untreated + Heat treated −Pichia farinose Untreated − Heat treated No data None − ++: developedstrong color, +: developed color, −: developed no color

As described above, the freeze-dried powders of the test strains(Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus natto, Bacillusthuringiensis, and Bacillus mycoides) showed meat color developmenteffect. In particular, Bacillus subtilis and Bacillus natto showed higheffect. In addition, the cultured bacterial cells were crushed by Frenchpress to obtain samples, and these samples showed similar meat colordevelopment effect (no data shown).

3. Meat Color Development Test 2

10 mg of the freeze-dried powder (crushed bacterial cells of Bacillussubtilis) was dissolved in 100 μL of 0.5 M phosphoric acid buffer (pH5.5). The solution was mixed with 10 mg of freeze-dried minced hampowder, 30 μL of 4% (w/v) myoglobin, 30 μL of 0.2 M NADH, and 400 μL ofsterilized water, and allowed to stand at room temperature for 30minutes. Subsequently, the reaction liquid was centrifuged at 15,000 rpmfor 15 minutes, and the supernatant was recovered. The absorptionspectrum of the resultant supernatant at wavelengths from 700 nm to400nm was measured using a spectrophotometer. As control test, a samplecontaining the powder solution boiled for 10 minutes was subjected tothe same test. The results are shown in FIG. 1. As is evident from theresults, the untreated sample showed absorption maxima at 545 nm and 580nm, indicating a red color.

4. Meat Color Development Test 3

According to the method described in 3, meat color development test wascarried out using the freeze-dried powders prepared in 1. As control, asample without the powder solution was subjected to the same test. Theabsorbance of the samples at a wavelength 580 nm is shown in FIG. 2. Theresult indicates that all the tested samples developed stronger colorthan the control even five days after.

5. Measurement Method for Metmyoglobin Reductase Activity

The metmyoglobin reductase activity was measured as follows. Firstly,the freeze-dried powders prepared in 1 were dissolved in water to make10 mg/mL solutions, and used as the enzyme solutions. Subsequently, 100μL of 0.1% (w/v) myoglobin and 150 μL of enzyme solution were added to200 μL of 0.1 M phosphoric acid buffer (pH 5.5), and preincubated at 30°C. for 5 minutes. Thereafter, 50 μL of 1 mM NADH was added, and thechange in the absorbance at a wavelength of 406 nm was measured for 5minutes. The activity was expressed in unit (U). Under the presentconditions, the amount of enzyme reducing substancially 1 μM ofmetmyoglobin in 1 minute was regarded as 1 U. For comparison, theenzymatic activity of the freeze-dried powder of Pichia farinosa strainIAM 12223 (=strain IFO0465, JCM1634) was also studied.

The measurement result is shown in FIG. 3. The freeze-dried powders withhigh meat color development effect exhibited high metmyoglobin reductaseactivity. The result indicates that the test strains (Bacillus subtilis,Bacillus amyloliquefaciens, Bacillus natto, Bacillus thuringiensis, andBacillus mycoides) produce metmyoglobin reductase, and the colordevelopment effect is brought about by the enzymatic action.

6. Purification of Metmyoglobin Reductase

The metmyoglobin reductase was purified as follows. The bacterial cellsof Bacillus subtilis obtained by culture described in 1 was crushed byFrench press, centrifuged, and then the supernatant was salted out withammonium sulfate. The supernatant was treated at 30% saturation andcollected, and treated at 70% saturation and centrifuged, and then theprecipitate was recovered. The precipitate was dissolved in a 20 mM KPB(pH=6.0) solution, dialyzed, and used as the ammonium sulfateprecipitation sample.

5 mL of the ammonium sulfate precipitation sample thus obtained wassubjected to DEAE chromatography under the following conditions (DEAEcolumn (HiTrap™ DEAE FF (5 mL); GE Healthcare)). As a result of this,the protein yield was 45.4%.

(DEAE Chromatography Conditions)

Carrier: DEAE I-0(5 mL)

Charge: ammonium sulfate precipitation sample (5 mL)

Buf A: 20 mM KPB (pH 6)

Buf B: 20 mM KPB (pH 6),1 M NaCl

Flow rate: 5 mL/minute

Fraction: 5 mL

Program:(1) Buf A washing 8 cv, (2) Buf B 10% washing 8 cv, (3) Buf Bgradient 30%/25 cv, (4) Buf B 100% 8 cv

FIG. 4 shows the elution pattern and metmyoglobin reductase activityobtained by the DEAE chromatography. The activity was measured by thedecrease of A406, which is the absorption maximum of metmyoglobin. Ofthe fractions shown in FIG. 4, DEAE Fr.No.61-63 with the highestactivity were subjected to hydroxyapatite chromatography (hydroxyapatitecolumn (1×5 cm) (TypeI 20 μm; Bio-Rad)) under the following conditions,thereby purifying the fractions. The protein yield of DEAE Fr. No.61-63was 98.5% as measured by hydroxyapatite chromatography.

(Hydroxyapatite Chromatography Conditions)

Carrier: hydroxyapatite (5 mL)

Charge: DEAE purified Fr. No. 61-63

Buf A: 5 mM KPB, 0.3 M NaCl (pH 6)

Buf B: 400 mM KPB, 0.3 M NaCl (pH 6)

Flow rate: 1 mL/minute

Fraction: 4 mL

Program: (1) Buf A washing 7 cv, (2) Buf B gradient 100%/20 cv, (3) BufB 100% 10 cv

FIG. 5 shows the elution pattern and metmyoglobin reductase activityobtained by hydroxyapatite chromatography of DEAE Fr. No. 61-63. Thehydroxyapatite Fr. No. 19, which showed the highest specific activity inFIG. 5, was dialyzed with 20 mM KPB (pH 6), and then subjected to gelfiltration chromatography under the following conditions (gel filtrationcolumn (Superdex TM75; GE Healthcare)).

(Gel Filtration Chromatography Conditions)

Carrier: Super dex 200 (120 mL)

Charge: Hydroxyapatite Fr.19

Buf A: 20 mM KPB (pH 6)

Flow rate: 1 mL/min

Fraction: 5 mL

FIG. 6 shows the elution pattern and metmyoglobin reductase activityobtained by gel filtration chromatography of the hydroxyapatite Fr. No.19. The gel filtration Fr. No. 12-16 containing the gel filtration Fr.No. 13-15, which showed particularly high specific activity in FIG. 6,was subjected to SDS-PAGE, and the band was confirmed (FIG. 7: gelfiltration Fr. No. 12-16). The gel filtration Fr. No. 13-15 in FIG. 7showed a single band.

FIG. 8 shows the specific activity of the fractions obtained in theabove purification processes (DEAE chromatography, hydroxyapatitechromatography, and gel filtration chromatography). DEAE. Fr. No. 61-63was subjected to gel filtration, and its specific activity was increasedto about 20.7 times.

7. Amino Acid Sequencing of Metmyoglobin Reductase

The single band obtained by SDS-PAGE shown in FIG. 7 was transferred toa PDVF membrane stained with Ponceau reagent, and cut out. TheN-terminal amino acid sequence was analyzed by a protein sequencer. As aresult of this, the N-terminal amino acid sequence was found to beVVGDFPIETDTLVIG (SEQ ID NO: 1). Furthermore, on the basis of the aminoacid sequence, the gene was searched from the Bacillus subtilis databasein BLAST. As a result of this, the gene was found to have 100% homologywith pdhD (SEQ ID NO: 2) coding dihydrolipoyl dehydrogenase (hereinafterreferred to as DLD). The amino acid sequence of DLD is shown in SEQ IDNO: 3.

8. Meat Color Development Test by DLD

Purified DLD was subjected to meat color development test. Using afreeze-dried powder sample of the purified DLD, meat samples wereprepared as described below (Table 3), stored at 4° C. overnight, andcompared (FIGS. 9 and 10).

TABLE 3 0.5M KPB 20 mM Minced 40 mg/mL (μL) NADH Freeze-dried pork (g)Mb (μL) (pH = 5.5) (μL) sample (mg) Meat 2 37.5 37.5 37.5 0 sample 1Meat 2 37.5 37.5 37.5 50 sample 2 Meat 2 37.5 37.5 37.5 20 sample 3 Meat2 37.5 37.5 37.5 10 sample 4 Meat 2 37.5 37.5 0 50 sample 5

FIG. 9 shows meat samples with different enzyme contents (meat samples 1to 4), indicating that DLD contributes to the improvement of color toneof meat. FIG. 10 shows the comparison of the color tone of meat with orwithout NADH (from left to right in this order, without freeze-driedpowder (meat sample 1), with NADH (meat sample 2), and without NADH(meat sample 5)). It was confirmed that color development issufficiently achieved even without NADH. The reason for this is likelythat the meat contains a sufficient amount of NADH.

9. Enzymological Properties of DLD

Using potassium ferricyanide, which has high affinity for the enzyme, asthe substrate, various properties were studied. Firstly, the substratereactivity of DLD was examined. The amount of enzyme was 15 μL, and rateassay (pH=6.0) was carried out for 30 seconds to give the substrateconcentrations (final concentrations) of 0.025, 0.05, 0.075, 0.1, 0.15,0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 1.0, 1.25, 1.5, 1.75, 2.0, and 2.5(mM). The molar absorptivity of potassium ferricyanide was 1.02×10³(M⁻¹.cm⁻¹), and the rate (v) was expressed in the unit of μM/minute. Theresults are shown in FIGS. 11 and 12. The [s]/v to [s] plot was preparedfrom the rising of the substrate-saturation curve shown in FIG. 11 (FIG.12), and the rate parameter (Kinetic parameter) was calculated (Km=0.19(mM), Vmax=26.2 (μM/minute)). For comparison, the Km value of Diapholasefrom other source having high affinity for potassium ferricyanide isshown in Table 4. Table 4 indicates that the DLD obtained from Bacillussubtilis has higher affinity for potassium ferricyanide than the DLDderived from C. kluyveri.

TABLE 4 Km Enzyme Source (mM) Literature lipoyl de- C. kluyveri 0.35Frank Petrat. et. al (2003) The hydrogenase Journal of BiologicalChemistry, 278, 46403-13 NADPH- C. kluyveri 0.13 Frank Petrat. et. al(2003) The glutathione Journal of Biological reductase Chemistry, 278,46403-13 NADH- C. kluyveri 0.58 Frank Petrat. et. al (2003) Thecytochrome Journal of Biological c reductase Chemistry, 278, 46403-13NADPH- C. kluyveri 0.88 Frank Petrat. et. al (2003) The cytochromeJournal of Biological P450 Chemistry, 278, 46403-13 reductase DiaphoraseB. 4.0 Tomokazu Matsue stearothermophilus et. al. (1990) Biochemica etBiophysica Acta, 1038, 29-38

The optimal pH of DLD was measured as follows. 50 μL of a 10-folddilution (50 mM) of any of 0.5 M pH buffers (citric acid buffer (pH=3.0to 6.0), potassium phosphate buffer (KPB) (pH=6.0 to 8.0), andTris-hydrochloric acid buffer (pH=8.0 to 10.0)), 50 μL of 10-folddilution (400 μM) of 4 mM potassium ferricyanide solution, 50 μL ofenzyme sample, and 250 μL of MilliQ water were incubated at 30° C. for 5minutes. Thereafter, 100 μL of 1 mM NADH solution was added, and thechange in the absorbance at A420 was monitored. The result is shown inFIG. 13.

Subsequently, the pH stability of DLD was studied as follows. 20 μL ofenzyme sample and 180 μL of any of 20 mM pH buffers (citric acid buffer(pH=3.0 to 6.0), potassium phosphate buffer (KPB) (pH=6.0 to 8.0),Tris-hydrochloric acid buffer (pH=8.0 to 10.0)) were mixed, the mixturewas allowed to stand and react for 30 minutes, and used as the pHtreatment sample solution. 150 μL of the pH treatment sample solutionthus obtained, 100 μL of 0.5 M citric acid buffer (pH=6.0), and 100 μLof MilliQ water were mixed, and stored on ice overnight. To 350 μL ofthe resultant storage solution, 50 μL of 4 mM potassium ferricyanide wasadded, and incubated at 30° C. for 5 minutes. Thereafter, 100 μL of 1 mMNADH solution was added, the change in the absorbance at A420 wasmonitored for 30 seconds at intervals of 1 second (The activity of thesample without pH treatment was taken as 100%). The result is shown inFIG. 14. The stability in the lower pH side was insufficient, but the pHin meat was from 5 to 6, so that the activity is likely sufficient inmeat.

The optimal temperature of DLD was measured as follows. 50 μL of 0.5 Mcitric acid buffer (pH 6), 50 μL of 2 mM potassium ferricyanide, 100 μLof MilliQ water, and 100 μL of 1 mM NADH were mixed, the mixture waspreincubated at different temperatures for 5 minutes, and mixed with 200μL of an enzyme sample. The change in the absorbance at A420 wasmeasured for 5 minutes, thereby confirming the reaction. The result isshown in FIG. 15. The result indicates that the optimal temperature is40° C., and deactivation occurs at 50° C.

Further, the thermal stability was measured as follows. The enzymesample, which had been treated in advance for 30 minutes at differenttemperatures (30° C., 40° C., and 60° C.), was cooled on ice, and thuspreparing treatment samples. 30 μL of the treatment sample was mixedwith 50 μL of 0.5 M citric acid buffer (pH 6.0), 50 μL of 2 mM potassiumferricyanide, and 270 μL of MilliQ water, incubated at 30° C. for 5minutes, and then mixed with 100 μL of 1 mM NADH solution. The change inthe absorbance at A420 was measured for 30 seconds at intervals of 1second. The result shown in FIG. 16 indicates that high activityremained even at 60° C.

Subsequently, reactivity for NADH and NADPH was compared. 50 μL of 0.5 MKPB (pH 5.5), 100 μL of purified DLD, and 100 μL of 0.1% Mb were mixed,the volume was adjusted to 400 μL with MilliQ water, and preincubated at30° C. for 5 minutes. Thereafter, 50 μL of 1 mM NaDH or NADPH was added,and A406 was monitored for 5 minutes. FIG. 17 shows the reactivity,taking the relative activity of NADH as 100(%). The result indicatesthat DLD has low reactivity for NADPH.

In addition, the influence of metal salts (metal cations) on theactivity of DLD was studied. 100 μL of 500 mM KPB (pH 5.5), 200 μL of0.1% (=13.4 μM) Mb, 150 μL of purified DLD, 10 μL of 100 mM cation(CaCl₂, MgCl₂, FeCl₃, SnCl₂, CuSO₄, FeCl₂, MnSO₄, CdCl₂, ZnCl₂, NaCl,KCl, SDS, or EDTA), and 100 μL of 1 mM NADH were mixed, and purifiedwater was added to the mixture to make the volume 1 mL. The solution wasused as the sample, and measured for the relative activity. Theimprovement in the activity was observed when Ca⁺, Mg²⁺, and K⁺ wereused (FIG. 18).

10. DLD Overexpression System

The DLD gene was obtained from Bacillus subtilis, and studied forestablishing an overexpression system using Escherichia coli.

10-1. Genome Extraction from Bacillus subtilis Strain 7417

The genome extraction from Bacillus subtilis strain 7417 was carried outas follows. Bacillus subtilis strain 7417 was inoculated in a liquidmedium (pH6.5) containing 0.5% peptone, 1.0% yeast extract, and 1.0%glucose, and cultured overnight at 30° C. under shaking at 300 rpm. Thegenome DNA was extracted from the culture thus obtained using QIA quick™Gel Extraction Test Kit (QIAGEN).

10-2. Amplification of DLD Gene by PCR

DLD gene amplification by PCR was carried out as follows. 5 μL of 10×buffer, 4 μL of dTNP, 1 μL of the genome of Bacillus subtilis, 5 μL eachof the following 10 μM primers (two types), and 0.1 μL of EX. Taq (DNApolymerase, Takara Bio Inc.) were mixed, and purified water was added tothe mixture to make the volume 50 μL. The primers were combined in twopatterns (patterns 1 and 2). The PCR reaction was carried out in twosteps. Firstly, as the step 1, the solution was denatured by heat at 98°C. for 30 seconds. Subsequently, as the step 2, the following cycle(heat denaturation: 98° C., 10 seconds, annealing: 46° C., 30 seconds,elongation reaction: 72° C., 90 seconds) was repeated 25 times, and thusobtaining a PCR product.

(Primer arrangement) Pattern 1 DLD-Nde1-FW: (SEQ ID NO: 4)GGCGTAATCATATGGTAGTAGGAG DLD-BamH1-RV: (SEQ ID NO: 5)GATAGGATCCTTATTTTACGATG Pattern 2 DLD-Nde1-FW: (SEQ ID NO: 6)GGCGTAATCATATGGTAGTAGGAG DLD-BamH1-Histag-RV: (SEQ ID NO: 7)GATAGGATCCTTAGTGGTGGTGGTGGTGGTGTTTT ACGATG

10-3. TA Cloning of DLD Gene

Subsequently, TA cloning of the DLD gene was carried out as follows. 3μL of the PCR product obtained by PCR was mixed with 5 μL of 2× Liationbuffer, 1 μL of pGEM-T easy vector, and 1 μL of T4 ligase. The mixturewas allowed to react overnight at 4° C., thoroughly poured intocompetent cell DH5α, subjected to heat shock at 42° C. for 30 seconds,and cooled on ice for 2 minutes. 150 μL of SOC culture medium was addedto this, and incubated at 37° C. for 20 minutes. The total amount wascultured on an LB/Amp culture plate, and thus obtaining colonies.

10-4. DLD Transformation

Subsequently, DLD was cloned into vector as follows. From the cultureobtained by TA cloning, plasmid was extracted using GenElute™ plasmidMiniprep Kit (SIGMA). The plasmid extract thus obtained was mixed with10× buffer, Nde I, and treated at 37° C. for 2 hours. Furthermore, 1 μLof BamH I was added, and treated at 37° C. for 1 hour to prepare a geneto be inserted. On the other hand, the pET20b vector was mixed with 1 μLof BamH I, and treated at 37° C. for 1 hour. In order to study theenzymatic activity of DLD with or without His-tag, samples were preparedas follows (unit is μL), and incubated at 16° C. for 30 minutes.Thereafter, the whole amount was added to competent cell DH5a, dissolvedon ice for 1 hour, and the cell was subjected to heat shock (42° C., 30seconds). The SOC culture medium was added to the cell, incubated at 37°C. for 20 minutes, and plated on an LB/amp culture medium.

10-5. Enzymatic Activity Measurement of Transformant

The activity of the transformant obtained as described above wasmeasured as follows. Five colonies of DLD(+)Histag/pET20b/BL21 and fourcolonies of DLD(−)Histag/pET20b/BL21 were transferred to LB/Amp culturemedia, and cultured at 30° C. overnight under shaking (preculture). 60μL of the preculture solution was inoculated in 3 mL of LB/Amp culturemedium, and cultured at 37° C. overnight under shaking (main culture).When the OD600 reached 0.4 to 0.5, IPTG was added to give the finalconcentration of 0.1 mM, and cultured at 30° C. for 4 hours undershaking. The bacterial cells thus obtained were collected, and suspendedin 50 mM Tris-HCl (pH=7.0). The suspension was crushed using a beadshocker (MULTI-BEADS SHOCKER, Yasui Kikai Corporation), centrifuged, andthen the supernatant was used as the sample.

Enzyme reaction (metmyoglobin reduction reaction) was carried out asfollows. 50 μL of the enzyme sample obtained by the above-describedmethod was mixed with 0.5 M KPB buffer (pH=5.5) and 50 μL of 0.1%metmyoglobin solution, and purified water was added to the mixture tomake the volume 225 μL. Subsequently, 25 μL of 1 mM NADH solution wasadded to initiate reaction, and the change in the absorbance at A406 wasmonitored for 10 minutes. At the same time, the protein amount wasdetermined by Bradford assay. The results are shown in FIGS. 19 and 20.

FIGS. 19 and 20 show the metmyoglobin reduction activity oftransformants without and with His-tag, respectively. For comparison,the data for the pET20b empty vector and IPTG vector are also shown.Both of them showed 10 times or more higher metmyoglobin reductionactivity than the empty vector. As shown by the comparison of FIGS. 19and 20, control by IPTG is likely not imposed.

11. Meat Color Development Activity of Recombinant DLD

The recombinant DLD obtained above was subjected to bead crushing,purified by an Ni-Sepharose column under the following conditions, anddianalyzed by 20 mM KPB (pH 6). After freeze-dried, it was used as thesample of meat color development test 1 and meat color development test2.

(Chromatography Conditions)

Carrier: Ni Sepharose (25 mL)

Sample: crushed supernatant about 20 mL

Bind Buf: 20 mM KPB, 0.3 M NaCl (pH 6)

Elute Buf: 20 mM KPB, 0.3M NaCl, 0.4 M imidazole (pH 6)

Flow rate: charge: 5 mL/minute, other: 10 mL/minute

Fraction:10 mL

Program: (1) Bind Buf washing 6 cv, (2) Elute Buf 10% washing 10 cv, (3)Elute Buf 100%/20 cv gradient, (4) Elute Buf washing 10 cv

In meat color development test 1, 2 g of minced pork was mixed with 37.5μL of 40 mg/mL (4%) myoglobin (SIGMA), 37.5 μL of 0.5 M KPB (pH=5.5),37.5 μL of 20 mM NADH, and 66 mg of freez-dried enzyme powder sample wasadded, and allowed to react at 4° C. overnight. For comparison, a samplewithout freeze-dried powder (negative control) were also prepared. Theresults are shown in FIG. 21. The left shows the negative control, andthe right shows the meat with the freez-dried powder. The change in thecolor tone was studied by visual observation and the RGB value of theimage, and the results indicate that the color tone of the meat was morereddened by the recombinant DLD.

In the meat color development test 2, 2 g of minced pork was mixed with37.5 μL of 0.5 M KPB (pH=5.5), 37.5 μL of 40 mg/mL (4%) myoglobin(SIGMA), 37.5 μL of 20 mM NADH, and a sample ((1) purified DLD alone (60mg), (2) food additive sodium nitrite alone (0.4% (w/w)=8 mg), (3)purified DLD+sodium nitrite ((1) and (2)), (4) mixture of (2) and zincgluconate (15 mg), (5) none) and allowed to react at 4° C. overnight(FIG. 22). Furthermore, the reaction product was heated at 65° C. for 85minutes, and the result is shown in FIG. 23. These samples shown inFIGS. 22 and 23 were studied by visual observation, and also by the RGBvalue of the images.

The color tone of the meat without heat treatment shown in FIG. 21 isdescribed below. Good red color tone was found in the meat containingDLD (samples (1) and (3)). In particular, the color tone of the sample(3) containing DLD and sodium nitrite was good. The sample (4)containing sodium nitrite and zinc gluconate developed a brown color.The sample (2) containing sodium nitrite alone showed the similar colortone to the sample (5) without additive.

The color tone of the meat after heat treatment shown in FIG. 22 isdescribed below. Good red color tone (white peach color) was found inthe meat containing sodium nitrite (samples (2), (3), and (4)). Inparticular, the sample (3) containing DLD and sodium nitrite developed astrong red and showed a good color tone. The sample (1) containing DLDalone had rather stronger redness than the sample (5) without additive,but was markedly browned in comparison with the samples (2) to (4).

12. Purification of Meat Color Developing Enzyme

Of the meat color developing enzymes derived from the bacterial cells ofBacillus subtilis, the meat color developing enzymes other than DLD werepurified. In the purification process, using the first half peaks ofDEAE chromatography (DEAR Fr. No. 27-35 shown in FIG. 4) as startingmaterials, phenyl chromatography, hydroxyapatite chromatography, and Cuaffinity chromatography were carried out.

10 mL of the first half fraction of DEAE (DEAE. Fr. No. 27 -3 5 shown inFIG. 4) was subjected to phenyl chromatography under the followingconditions (phenyl column (HiTrap™ Phenyl HP (5 mL); GE Healthcare)).The elution pattern and metmyoglobin reductase activity obtained by thephenyl chromatography are shown in

FIG. 24. Of these fractions, the phenyl. Fr. No. 26-31 with the highestactivity were dialyzed with 5 mM KPB (pH 6), and subjected tohydroxyapatite chromatography.

(Phenyl Chromatography Conditions)

Carrier: Phenyl HP (5 mL)

Sample: DEAE first half Fr. (100210) UF 10 mL

Buf A: 20 mM KPB, 30% saturated ammonium sulfate (pH 6)

Buf B: 20 mM KPB (pH 6)

Flow rate: 5 mL/minute

Fraction: 5 mL

Program: (1) Buf A washing 5 cv, (2) Buf B gradient 100%/20 cv, (3) BufB 100% washing 5 cv

The phenyl. Fr. No. 26-31 obtained by the above-described phenylchromatography were subjected to hydroxyapatite chromatography under thefollowing conditions. FIG. 25 shows the elution pattern and metmyoglobinreductase activity obtained in the hydroxyapatite chromatography.

(Hydroxyapatite Chromatography Conditions)

Carrier: hydroxyapatite (5 mL)

Charge: phenyl purified Fr. No. 26-31

Buf A: 5 mM KPB, 0.3 M NaCl (pH 6)

Buf B: 400 mM KPB, 0.3 M NaCl (pH 6)

Flow rate: 2 mL/minute

Fraction: 5 mL

Program:(1) Buf A washing 5 cv, (2) Buf B gradient 100%/25 cv, (3) Buf B100% 6 cv

Of the fractions obtained in the hydroxyapatite chromatography, thehydroxyapatite Fr. No. 16 with the highest activity was dialyzed with 20mM KPB and 0.3 M NaCl (pH 6), and subjected to Cu affinitychromatography under the following conditions using a Cu affinitycolumn. FIG. 26 shows the elution pattern and metmyoglobin reductaseactivity obtained in the Cu affinity chromatography. Of these fractionsobtained in the Cu affinity chromatography, the Cu. Fr. No. 10, 13 withthe highest activity were subjected to SDS-PAGE. The results are shownin FIG. 27.

(Cu Affinity Chromatography Conditions)

Carrier: Cu2+ HP (1 mL)

Charge: hydroxyapatite-Fr. 16

Buf A: 20 mM KPB, 0.3 M NaCl (pH 6)

Buf B: 20 mM KPB, 0.3 M NaCl, 0.4 M imidazole (pH 6)

Flow rate: 1 mL/minute

Fraction: 2 mL

Program: (1) Buf A washing 6 cv, (2) Buf B gradient 10%/20 cv, (3) Buf Bwashing 10 cv

FIG. 27 shows the result of SDS-PAGE on the hydroxyapatite Fr. No. 13-17obtained in the hydroxyapatite chromatography, and the Cu. Fr. No. 8-15obtained in the Cu affinity chromatography. Of these fractions, the Cu.Fr. No. 10 obtained in Cu affinity chromatography showed two main bands.The fraction was subjected to the N-terminal amino acid sequenceanalysis by the above-described method; the protein with a highermolecular weight was MGNTRKKVSVI (SEQ ID NO: 8), and the protein with alower molecular weight was MTNTLDVLKA (SEQ ID NO: 9). On the basis ofthe N-terminal amino acid sequence, the protein with a higher molecularweight was subjected to BLAST search, and found to have 100% homologywith mdh (SEQ ID NO: 10) coding malate dehydrogenase (MDH). The proteinwith a lower molecular weight showed 100% homology with yodC (SEQ ID NO:11) coding putative NAD(P)H nitroreductase (yodC). The amino acidsequence of yodC is shown in SEQ ID NO: 12. The Cu. Fr. No. 13 wasassumed to be identical with dihydrolipoyl dehydrogenase, on the basisof its molecular weight.

16. Construction of Overexpression System of Meat Color DevelopingEnzymes (MDH, yodC)

Genes were obtained from Bacillus subtilis and Bacillus natto, and theoverexpression system for MDH and yodC was constructed. Primers weremade from the gene information of Bacillus subtilis and Bacillus natto,and subjected to PCR to cut out the genes. Using pET20b as the vectorand BL21 (DE3 pLysS) as the host, 6× His-tag was added to the C-terminalof the enzymes for expression. The expression was confirmed by thefollowing culture. The colonies of each enzyme (MDH oryodC)(+)Histag/pET20b/BL21 were taken, and cultured overnight at 30° C.under shaking at 300 rpm (preculture). A portion equivalent to 2% of thepreculture solution was inoculated in 10 mL of LB/Amp culture medium,and IPTG was added to give the final concentration of 0.5 mM when the ODreached about 0.5 to 0.7, and cultured for 4 hours at 37° C. undershaking at 300 rpm (main culture). The bacterial cells thus obtainedwere collected, and suspended in 50 mM Tris-HCl (pH=7.0). The suspensionwas subjected to bead crushing, centrifuged, and the supernatant wasused as the sample.

The activity of the sample obtained above was confirmed. 150 μL of theenzyme sample was mixed with 50 μL of 0.1% (w/w) metmyoglobin and 0.5 MKPB (pH=5.5), 25 μL of 1 mM NADH was added to the mixture to initiatereaction, and the change in the absorbance at A406 was monitored for 10minutes. As shown in FIG. 28, the activity of yodC was confirmed, butthe activity of MDH was not found. Therefore, in order to confirm thepresence or absence of expression, bands were observed by SDS-PAGE (FIG.29). Thick bands were found at the corresponding size for both MDH andyodC.

17. Color Development Test on Purified Meat Color Developing Enzymes(MDH, yodC)

The recombinant yodC (Bacillus subtilis) and MDH (Bacillus subtilis)obtained by the above-described method were purified by Ni-Sepharosecolumn under the following conditions, and dialyzed by 20 mM KPB (pH 6).The dialyzed sample was freeze-dried, and the enzyme powders of therecombinant yodC and MDH were obtained.

(Chromatography Conditions)

Carrier: Ni Sepharose (25 mL)

Sample: crushed supernatant about 20 mL

Bind Buf: 20 mM KPB, 0.3 M NaCl (pH 6)

Elute Buf: 20 mM KPB, 0.3 M NaCl, 0.4 M imidazole (pH 6)

Flow rate: charge: 5 mL/minute, other: 10 mL/minute

Fraction: 10 mL

Program: (1) Bind Buf washing 6 cv, (2) Elute Buf 10% washing 10 cv, (3)Elute Buf 100%/20 cv gradient, (4) Elute Buf washing 10 cv

Using the enzyme powders thus obtained, meat color development test wascarried out. In the meat color development test, 2 g of minced pork wasmixed with 37.5 μL of 40 mg/mL (4%) of myoglobin (SIGMA), 37.5 μL of 0.5M KPB (pH=5.5), 37.5 μL of 20 mM NADH, and sample ((1) control (withoutaddition), (2) yodC (16 mg=20 U), (3) boiled yodC, (4) MDH (16 mg), (5)boiled MDH), and allowed to react at 4° C. overnight. In order toconfirm deactivation by heat treatment (100° C., 30 minutes), thesamples (3) and (5) were provided. The results are shown in FIG. 30.Meat color development effect was found in yodC, but not found in MDH.

18. Study of Enzymological Properties of yodC

The enzymological properties of yodC were studied. In the same mannerfor DLD, the optimal pH (FIG. 31), pH stability (FIG. 32), optimaltemperature (FIG. 33), thermal stability (FIG. 34), reactivity for NADPH(FIG. 35), and the influence of the metal salt (metal cation) onactivity (FIG. 36) were studied. The optimal pH is in the vicinity of pH6.0 as is the case with DLD, so that the application to meat will offerno problem. In addition, yodC was stable over a wide pH range. Regardingthe optimal temperature, yodC favorably acts at low temperatures near20° C., so that yodC is regarded as suitable for the application tomeat. Also in the heat stability test, the activity was maintained up to40° C. In the coenzyme specificity test, higher activity was achieved inthe case using NADPH than the case using NADH. For metal cations,improvement in enzymatic activity was found when Mg, Na, or K was added.In addition, substrate reactivity was also studied. Under the sameconditions for DLD, reactivity for potassium ferricyanide, andreactivity for myoglobin were studied. The results including the data ofDLD are shown in FIGS. 37 and 38. In comparison with DLD, yodC showedabout 2.6 times and 22 times higher reactivity for potassiumferricyanide and myoglobin, respectively.

INDUSTRIAL APPLICABILITY

The reducing agent of the present invention is particularly useful as acolor tone improver for meat or processed meat. The reducing agent ofthe present invention develops the color of meat without using a colordevelopment agent such as a nitrite, and thus allows the production ofprocessed meat product with high commercial value.

The present invention will not be limited to the above-describedembodiments and examples of the invention. The present inventionincludes various modifications which can be readily made by thoseskilled in the art without departing from the scope of claims.

The contents of the articles, unexamined patent publications, and patentapplications specified herein are hereby incorporated herein byreference.

[Sequence List Free Text]

SEQ ID NO: 4: explanation of artificial arrangement: primer DLD-Nde1-FW

SEQ ID NO: 5: explanation of artificial arrangement: primer DLD-BamH1-RV

SEQ ID NO: 6: explanation of artificial arrangement: primer DLD-Nde1-FW

SEQ ID NO: 7: explanation of artificial arrangement: primerDLD-BamH1-Histag-RV

1. A reducing agent comprising a heme reductase derived from amicroorganism belonging to the genus Bacillus.
 2. The reducing agent ofclaim 1, wherein the heme is the heme of metmyoglobin.
 3. The reducingagent of claim 1, wherein the heme is the heme of methemoglobin.
 4. Thereducing agent of claim 1, which is composed of crushed bacterial cellsof a microorganism belonging to the genus Bacillus.
 5. The reducingagent of claim 1, wherein the microorganism belonging to the genusBacillus is a microorganism selected from the group consisting ofBacillus subtilis, Bacillus amyloliquefaciens, Bacillus natto, Bacillusthuringiensis, and Bacillus mycoides.
 6. The reducing agent of claim 1,wherein the heme reductase is dihydrolipoyl dehydrogenase ornitroreductase.
 7. The reducing agent of claim 1, which containsdihydrolipoyl dehydrogenase and nitroreductase as the heme reductase. 8.The reducing agent of claim 6, wherein the amino acid sequence of thedihydrolipoyl dehydrogenase includes the amino acid sequence of SEQ IDNO: 3, and the amino acid sequence of the nitroreductase includes theamino acid sequence of SEQ ID NO:
 12. 9. The reducing agent of claim 6,wherein the dihydrolipoyl dehydrogenase and nitroreductase arerecombinant proteins.
 10. A color tone improver composed of the reducingagent of claim
 1. 11. A color tone improver composed of the reducingagent of claim 1 and a substance which substitutes iron in the hemegroup of myoglobin with zinc.
 12. The color tone improver of claim 11,wherein the substance is ferrochelatase.
 13. The color tone improver ofclaim 10, which is used for improvement of the color tone of meat orprocessed meat.
 14. A color tone improver for meat or processed meatcomprising dihydrolipoyl dehydrogenase and/or nitroreductase.
 15. Thecolor tone improver of claim 10, which improves the color tone by colordevelopment action, color development acceleration action, and/or colorfading preventive action.
 16. A medicine comprising the reducing agentof claim
 1. 17. The medicine of claim 16, which is an oral preparation.18. The medicine of claim 16, which is a parenteral preparation.
 19. Amethod for producing a reducing agent comprising the following steps (1)and (2): (1) a step of culturing a microorganism belonging to the genusBacillus producing a heme reductase under conditions suitable for theproduction of the enzyme; and (2) a step of recovering the enzyme fromthe culture.
 20. The production method of claim 19, wherein the step (2)includes the following steps: (2-1) a step of collecting bacterial cellsfrom the culture; and (2-2) a step of preparing crushed bacterial cells.21. The production method of claim 19, wherein the heme is the heme ofmetmyoglobin.
 22. The production method of claim 19, wherein the heme isthe heme of methemoglobin.
 23. The production method of claim 19,wherein the microorganism belonging to the genus Bacillus is selectedfrom the group consisting of Bacillus subtilis, Bacillusamyloliquefaciens, Bacillus natto, Bacillus thuringiensis and Bacillusmycoides.
 24. A color tone improvement method, comprising subjectingmeat or processed meat to the action of the color tone improver of claim10.
 25. A color tone improvement method, comprising subjecting meat orprocessed meat to the action of crushed bacterial cells of amicroorganism belonging to the genus Bacillus selected from the groupconsisting of Bacillus subtilis, Bacillus amyloliquefaciens, Bacillusnatto, Bacillus thuringiensis, and Bacillus mycoides.
 26. A prophylacticor therapeutic method using the reducing agent of claim 1 for a diseaseassociated with or caused by one or more clinical conditions or symptomsselected from blood circulation disorder and hypoxia or hypoxemia.